Temperature-responsive polymer/polymer complex

ABSTRACT

The problem of the invention is to find a polymer mixture which forms an inter-polymer complex by being responsive to temperature even under neutral to alkaline conditions. According to the invention, a temperature responsive inter-polymer complex capable of showing thermal responsiveness in an aqueous solution, which contains a poly-N-acetylacrylamide or polyvinyl alcohol and polyethylene glycol, polyacrylamide or polymethacylamide, is applied to separating agents, immobilized enzymes, denatured protein modifiers, separation method or concentration of microorganisms, purification or concentration of nucleic acids, drug-releasing microcapsules and the like.

TECHNICAL FIELD

This invention relates to a temperature responsive inter-polymer complexwhich can be applied to separating agents, immobilized enzymes,denatured protein modifiers, separation method or concentration ofmicroorganisms, purification or concentration of nucleic acids,drug-releasing microcapsules and the like.

BACKGROUND ART

Polyethylene oxide (PEO), polyacrylamide (PAAm), polyacrylic acid (PAAc)or polymethacrylic acid (PMAAc), which is a water-soluble polymer,dissolves in water in an aqueous solution within an optional temperaturerange. However, it has been described by Yoshihito Nagata and TasukuSaito in Nippon Kagaku Kaishi (Journal of Japanese Chemical Society), 1,171 (1976) that a mixed aqueous solution of PEO with PAAc or PMAAc formsan inter-polymer complex under an acidic condition, and the complexshows lower critical solution temperature (LCST) in the aqueoussolution.

On the other hand, it has been described by D. J. Eustance et al. in J.Appl. Polym. Sci., 35, 707 (1988) that a mixed aqueous solution of PAAmwith PAAc forms an inter-polymer complex under an acidic condition, andthe complex shows upper critical solution temperature (UCST).

However, the polymers shown in the above form inter-polymer complexeswith polyacrylic acid (PAAc) or polymethacrylic acid (PMAAc), which arepolyelectrolytes, but the inter-polymer complexes are not formed underneutral to alkaline conditions due to dissociation of carboxyl groups ofthe polyelectrolytes, so that the application range has a limitation.

Accordingly, an object of the invention is to find a technique by whichinter-polymer complexes can be formed by being responsive to temperatureeven under neutral to alkaline conditions.

DISCLOSURE OF THE INVENTION

This time, we have made an attempt to form an inter-polymer complexbetween nonionic polymers and found as a result that an inter-polymercomplex can be formed between nonionic water-soluble polymers, thusresulting in the accomplishment of the invention. That is, the inventionis comprised of the following constructions.

-   (1) A temperature responsive inter-polymer complex capable of    showing thermal responsiveness in an aqueous solution, which    contains a poly-N-acetylacrylamide and polyethylene glycol.-   (2) A temperature responsive inter-polymer complex capable of    showing thermal responsiveness in an aqueous solution, which    contains a poly-N-acetylacrylamide and polyacrylamide or    polymethacrylamide.-   (3) A temperature responsive inter-polymer complex capable of    showing thermal responsiveness in an aqueous solution, which    contains polyvinyl alcohol and polyethylene glycol.-   (4) A temperature responsive inter-polymer complex capable of    showing thermal responsiveness in an aqueous solution, which    contains polyvinyl alcohol and polyacrylamide or polymethacrylamide.-   (5) The temperature responsive inter-polymer complex described in    the aforementioned (1) or (2), wherein the poly-N-acetylacrylamide    is a copolymer of N-acetylacrylamide with a hydrophilic or    hydrophobic monomer.

Since the aforementioned inter-polymer complex of the invention can showtemperature response even under neutral to alkaline conditions, itsapplication range is sharply extended so that, e.g., by fixing a ligandhaving the ability to recognize molecules, it can be broadly applied toseparating agents, assay reagents, immobilized enzymes, denaturedprotein modifiers, separation method or concentration of microorganisms,purification or concentration of nucleic acids, drug-releasingmicrocapsules and the like. Thus, the invention is further comprised ofthe following constructions.

-   (6) The temperature responsive inter-polymer complex described in    any one of the aforementioned (1) to (5), wherein a ligand having    the ability to recognize molecules is fixed to at least one of the    poly-N-acetylacrylamide, polyvinyl alcohol, polyethylene glycol,    polyacrylamide and polymethacrylamide.-   (7) A separating agent which uses the temperature responsive    inter-polymer complex described in the aforementioned (6).

BEST MODE FOR CARRYING OUT THE INVENTION

More illustratively, a mixture of the poly-N-acetylacrylamide andpolyethylene glycol, or a mixture of a copolymer of N-acetylacrylamidewith a hydrophilic or hydrophobic monomer and polyacrylamide orpolymethacrylamide, according to the invention, shows upper criticalsolution temperature (UCST) in an aqueous solution. That is, it forms aninter-polymer complex at a temperature of equal to or lower than theUCST. Particularly, it has a coacervation ability.

Also, a mixture of the poly-N-acetylacrylamide and polyacrylamide orpolymethacrylamide shows lower critical solution temperature (LCST) inan aqueous solution. That is, it forms an inter-polymer complex at atemperature of equal to or higher than the LCST.

In addition, a mixture of polyvinyl alcohol and polyacrylamide orpolymethacrylamide, or a mixture of polyvinyl alcohol and polyethyleneglycol, shows both of UCST and LCST in an aqueous solution. That is, itforms an inter-polymer complex at a temperature of equal to or lowerthan UCST and of equal to or higher than LCST and has a coacervationability.

As the aforementioned hydrophilic monomer to be copolymerized withN-acetylacrylamide, acrylamide, methacrylamide, N-methylacrylamide,N-ethylacrylamide, N,N-dimethylacrylamide, allyl alcohol and the likecan be exemplified. Also, as the hydrophobic monomer to be copolymerizedwith N-acetylacrylamide, an acrylic acid ester, a methacrylic acidester, styrene, an N-alkyl (preferably an alkyl having 3 or more carbonatoms) acrylamide, vinyl chloride, ethylene, propylene and the like canbe exemplified.

It is desirable that the aforementioned hydrophilic or hydrophobicmonomer is used at a copolymerization ratio of from 0.5 to 80%,particularly at a copolymerization ratio of from 5 to 70%, based onN-acetylacrylamide.

According to the invention, the LCST (lower critical solutiontemperature) means a temperature condition under which a mixture keeps adissolved state at a specified temperature or less but aggregates bybecoming insoluble in its solution at the specified temperature or more,and the UCST (upper critical solution temperature) means a temperaturecondition under which a mixture keeps a dissolved state at a specifiedtemperature or more but aggregates by becoming insoluble in its solutionat the specified temperature or less. That is, e.g., the term “a polymermixture shows lower critical solution temperature (LCST) in an aqueoussolution” means that it is uniformly dispersed in a solution at aspecified temperature (LCST) or less but aggregates by forming aninter-polymer complex when the solution temperature is increased to thespecified temperature (LCST) or more.

Molecular weight of the inter-polymer complex obtained by the inventionis not particularly limited and can be optionally set in response to itsuse and the like. Generally, a range of from several hundred to severalmillion is appropriate. Also, molecular weight of each polymerconstituting the inter-polymer complex is not particularly limited too,but it is desirable that difference in the molecular weight betweenrespective polymers constituting the inter-polymer complex is small.When difference in the molecular weight is small, a temperatureresponsive inter-polymer complex having good thermal responsiveness withless transition temperature widths of UCST and LCST is formed.

In addition, concentration of each polymer dissolved in an aqueoussolution can also be optionally set in response to its use. A goodtemperature response can be obtained preferably at 0.1% or more. Also,in the case of an inter-polymer complex showing UCST for example, higherconcentration of polymers in the aqueous solution causes a tendency ofshifting the UCST to high temperature side.

Since the temperature responsive polymer mixture of the invention showsUCST or LCST even under neutral to alkaline conditions, variousapplications are possible. Particularly, since a ligand having theability to recognize molecules can be fixed to any one of the polymersconstituting the aforementioned temperature responsive polymer mixture,it can be broadly applied thereby to separating agents, assay reagents,immobilized enzymes, denatured protein modifiers, separation method orconcentration of microorganisms, purification or concentration ofnucleic acids, drug-releasing microcapsules and the like.

As the ligand having the ability to recognize molecules, antigens,antibodies, biotin and the like can be exemplified.

EXAMPLES

The invention is described further in detail in the following withreference to examples, but the invention is not restricted by theseexamples. In this connection, regarding the measurement of UCST and LCSTin aqueous solutions, they were measured using transmittance of avisible light (550 nm). Also, the UCST or LCST was defined as atemperature for 50% transmittance of the visible light.

Example 1

(Inter-polymer Complex 1 of Poly-N-acetylacrylamide and PEG)

When 800 mg of poly-N-acetylacrylamide having a molecular weight ofabout 13,000 was mixed with 400 mg of polyethylene glycol having amolecular weight of about 8,500 in 10 g of purified water, aninter-polymer complex was formed showing cloudiness. When thisinter-polymer complex was observed under an optical microscope,coacervates each having a diameter of about 1 micron were formed.

The thus obtained coacervates showed UCST in the aqueous solution, andthe temperature was 51° C. both at the time of temperature up andtemperature down. In this connection, measurement of UCST was carriedout after adjusting pH of the aqueous solution to 7.4.

Example 2

(Inter-polymer Complex 2 of Poly-N-acetylacrylamide and PEG)

When 800 mg of poly-N-acetylacrylamide having a molecular weight ofabout 13,000 was mixed with 800 mg of polyethylene glycol having amolecular weight of about 8,500 in 10 g of purified water, aninter-polymer complex in the form of coacervates was formed.

The thus obtained coacervates showed UCST in the aqueous solution, andthe temperature was 76° C. at the time of temperature up and 80° C. atthe time of temperature down. In this connection, measurement of UCSTwas carried out after adjusting pH of the aqueous solution to 7.4.

Example 3

(Inter-polymer Complex 3 of Poly-N-acetylacrylamide and PEG)

When 1.4 g of poly-N-acetylacrylamide having a molecular weight of about13,000 was mixed with 320 mg of polyethylene glycol having a molecularweight of about 3,000 in 10 g of purified water, an inter-polymercomplex in the form of coacervates was formed.

The thus obtained coacervates showed UCST in the aqueous solution, andthe temperature was 23° C. at the time of temperature up and 10° C. atthe time of temperature down. In this connection, measurement of UCSTwas carried out after adjusting pH of the aqueous solution to 7.4.

Example 4

(Inter-polymer Complex of Poly-N-acetylacrylamide and Polyacrylamide)

When 1.3 g of poly-N-acetylacrylamide having a molecular weight of about13,000 was mixed with 1.4 g of polyacrylamide having a molecular weightof about 14,000 in 10 g of purified water, an inter-polymer complex wasformed.

The thus obtained inter-polymer complex showed LCST in the aqueoussolution, and the temperature was 23° C. both at the time of temperatureup and temperature down. In this connection, measurement of LCST wascarried out after adjusting pH of the aqueous solution to 7.4.

Example 5

(Inter-polymer Complex 1 of N-acetylacrylamide-acrylamide (1:2)Copolymer and Polyacrylamide)

When 200 mg of an N-acetylacrylamide-acrylamide (1:2) copolymer having amolecular weight of about 340,000 was mixed with 8 mg of polyacrylamidehaving a molecular weight of about 10,000 in 10 g of purified water, aninter-polymer complex in the form of coacervates was formed.

The thus obtained coacervates showed UCST in the aqueous solution, andthe temperature was 35° C. both at the time of temperature up andtemperature down. In this connection, measurement of UCST was carriedout after adjusting pH of the aqueous solution to 7.4.

Example 6

(Inter-polymer Complex 2 of N-acetylacrylamide-acrylamide (1:2)Copolymer and Polyacrylamide)

When 400 mg of an N-acetylacrylamide-acrylamide (1:2) copolymer having amolecular weight of about 340,000 was mixed with 16 mg of polyacrylamidehaving a molecular weight of about 10,000 in 10 g of purified water, aninter-polymer complex in the form of coacervates was formed.

The thus obtained coacervates showed UCST in the aqueous solution, andthe temperature was 66° C. both at the time of temperature up andtemperature down. In this connection, measurement of UCST was carriedout after adjusting pH of the aqueous solution to 7.4.

Example 7

(Inter-polymer Complex 3 of N-acetylacrylamide-acrylamide (1:2)Copolymer and Polyacrylamide)

When 400 mg of an N-acetylacrylamide-acrylamide (1:2) copolymer having amolecular weight of about 340,000 was mixed with 16 mg of polyacrylamidehaving a molecular weight of about 10,000 in 10 g of PBS saline buffersolution, an inter-polymer complex in the form of coacervates wasformed.

The thus obtained coacervates showed UCST in the aqueous solution, andthe temperature was 34° C. both at the time of temperature up andtemperature down. In this connection, measurement of UCST was carriedout after adjusting pH of the aqueous solution to 7.4.

Example 8

(Inter-polymer Complex of Polyvinyl Alcohol and Polyethylene Glycol)

When 1 g of polyvinyl alcohol having a molecular weight of about 16,000was mixed with 500 mg of polyethylene glycol having a molecular weightof about 8,000 in 10 g of purified water, an inter-polymer complex inthe form of coacervates was formed.

The thus obtained coacervates showed UCST in the aqueous solution, andthe temperature was 42° C. both at the time of temperature up andtemperature down. In this connection, measurement of UCST was carriedout after adjusting pH of the aqueous solution to 7.4.

Example 9

(Inter-polymer Complex of Polyvinyl Alcohol (80% Hydrolysate) andPolyacrylamide)

When 400 mg of polyvinyl alcohol (80% hydrolysate) having a molecularweight of about 10,000 was mixed with 400 mg of polyacrylamide having amolecular weight of about 10,000 in 10 g of PBS saline buffer solution,an inter-polymer complex in the form of coacervates was formed.

The thus obtained coacervates showed LCST and UCST in the aqueoussolution. The LCST was 35° C. both at the time of temperature up andtemperature down, and the UCST was 84° C. both at the time oftemperature up and temperature down. In this connection, measurement ofLCST was carried out after adjusting pH of the aqueous solution to 7.4.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one skilled in theart that various changes and modifications can be made therein withoutdeparting from the spirit and scope thereof.

This application is based on Japanese Patent Application No. 2000-252526applied on Aug. 23, 2000, the entire contents thereof being herebyincorporated by reference.

INDUSTRIAL APPLICABILITY

The polymer mixture of the invention forms a polymer complex by beingresponsive to temperature and shows UCST and/or LCST in an aqueoussolution even under neutral to alkaline conditions.

Thus, by fixing a ligand having the ability to recognize molecules toanyone of components of the polymer mixture, it can be broadly appliedto various separating agents, assay reagents, denatured proteinmodifiers, agents for purifying nucleic acids, drug-releasingmicrocapsules and the like.

1. A temperature responsive inter-polymer complex capable of showingthermal responsiveness in an aqueous solution, which comprises apoly-N-acetylacrylamide and polyethylene glycol, wherein a ligand havingthe ability to recognize molecules is fixed to at least one of thepoly-N-acetylacrylamide and polyethylene glycol.
 2. A separating agentwhich uses the temperature responsive inter-polymer complex according toclaim
 1. 3. A temperature responsive inter-polymer complex capable ofshowing thermal responsiveness in an aqueous solution, which comprises apoly-N-acetylacrylamide and polyacrylamide or polymethacrylamide,wherein the poly-N-acetylacrylamide is a copolymer of N-acetylacrylamidewith a hydrophilic or hydrophobic monomer.
 4. A separating agent whichuses the temperature responsive inter-polymer complex according to claim3.
 5. A temperature responsive inter-polymer complex capable of showingthermal responsiveness in an aqueous solution, which comprises apoly-N-acetylacrylamide and polyacrylamide or polymethacrylamide,wherein a ligand having the ability to recognize molecules is fixed toat least one of the poly-N-acetylacrylamide, polyacrylamide andpolymethacrylamide.
 6. A separating agent which uses the temperatureresponsive inter-polymer complex according to claim
 5. 7. A temperatureresponsive inter-polymer complex capable of showing thermalresponsiveness in an aqueous solution, which comprises polyvinyl alcoholand polyethylene glycol, wherein a ligand having the ability torecognize molecules is fixed to at least one of the polyvinyl alcoholand polyethylene glycol.
 8. A separating agent which uses thetemperature responsive inter-polymer complex according to claim
 7. 9. Atemperature responsive inter-polymer complex capable of showing thermalresponsiveness in an aqueous solution, which comprises polyvinyl alcoholand polyacrylamide or polymethacrylamide, wherein a ligand having theability to recognize molecules is fixed to at least one of the polyvinylalcohol, polyacrylamide and polymethacrylamide.
 10. A separating agentwhich uses the temperature responsive inter-polymer complex according toclaim
 9. 11. A temperature responsive inter-polymer complex capable ofshowing thermal responsiveness in an aqueous solution, which comprises apoly-N-acetylacrylamide and polyethylene glycol, wherein thepoly-N-acetylacrylamide is a copolymer of N-acetylacrylamide with ahydrophilic or hydrophobic monomer, and wherein a ligand having theability to recognize molecules is fixed to at least one of thepoly-N-acetylacrylamide and polyethylene glycol.
 12. A separating agentwhich uses the temperature responsive inter-polymer complex according toclaim 11.